The invention relates to organic compounds acting as chemokine receptor ligands, including antagonists of CXC chemokine receptor-4 (CXCR-4) and therapeutic uses thereof, such as in the treatment of hematopoietic cells and in the treatment of chemokine or chemokine receptor mediated diseases.
Cytokines are soluble proteins secreted by a variety of cells including monocytes or lymphocytes that regulate immune responses. Chemokines are chemotactic cytokines that belongs to a large family of chemoattractant molecules involved in the directed migration of immune cells. They regulate a variety of biological responses and promote the recruitment of multiple lineages of leukocytes and lymphocytes to a specific body organ tissues. The functional role generally assigned to chemokines in the immune process is to elicit mobilization of immune cells against pathogenic organisms by direct recruitment and activation. Based on their structural similarity, chemokines may be subdivided into four subfamilies, CXC, CC, C and CX3C, depending on the position of their first two cysteine residues. SDF-1 belongs to the CXC chemokine family. It exerts its biological activities by binding to a specific cell surface receptor, CXC Chemokine receptor 4 (CXCR-4). In human, CXC chemokine genes are clustered on chromosome 4 (with the exception of SDF-1 gene, which has been localized to chromosome 10) and CC chemokine genes are clustered on chromosome 17.
The molecular targets for chemokines are cell surface receptors CXCR-4, one such receptor is a G protein coupled 7 transmembrane protein, and was previously called LESTR. Majority of chemokine receptors identified to date bind several distinct chemokines at high affinity, with the exception of CXCR-4, which binds only SDF-1. SDF-1 is thought to be the natural ligand for CXCR-4. CXCR-4 is widely expressed on cells of hematopoietic origin, and is a major co-receptor with CD4+ for human immunodeficiency virus 1 (HIV-1). CXCR-4 was found to be overexpressed in glioblastoma multiforme tumor tissue (GMTT), as compared to normal brain tissue (NBT). Expression analysis indicated that CXCR-4 is overexpressed in 57% of the primary glioblastoma tissues and in 8% of the glioblastoma cell lines. Gene-specific RT-PCR analysis indicated that the CXCR-4 gene is overexpressed in several malignant glioma tissues, breast tumor tissues and cell lines. Northern blot analysis indicated expression of CXCR-4 at high levels in certain leukemias, uterine cancer, and Burkitt""s lymphoma cell lines. The occipital and temporal lobe showed high levels of CXCR-4 in normal human brain. In adult mouse, CXCR-4 is expressed only in brain, spinal cord, bone marrow, and pituitary gland. Antisense CXCR-4 overexpression in glioblastoma cells caused inhibition of cell proliferation and induction of cellular differentiation in vitro. These findings indicate that CXCR-4 expression may play an important role during embryonic development and also in the genesis of human gliomas; that CXCR-4 plays an important role in the tumorigenic properties of brain, breast, and other tumor types. Its unique expression during mouse development also indicates that CXCR-4 plays an important role in the normal function of brain, spinal cord, and bone marrow during development.
SDF-1 gene occurs in two alternative splicing variants, producing SDF-1 alpha and SDF-1 beta (together referred to herein as SDF-1). The native and genomic amino acid sequences of SDF-1 alpha and SDF-1 beta have been determined .
SDF-1 is functionally distinct from other chemokines in that it is reported to have a fundamental role in the trafficking, exporting and homing of bone marrow progenitor cells. It is also structurally distinct in that it has only about 22% amino acid sequence identity with other CXC chemokines. SDF-1 appears to be produced constitutively by several cell types, and particularly high levels are found in bone-marrow stromal cells. A basic physiological role for SDF-1 is implied by the high level of conservation of the SDF-1 sequence between species. In vitro, SDF-1 stimulates chemotaxis of a wide range of cells including monocytes and bone marrow derived progenitor cells. It also stimulates a high percentage of resting and activated T-lymphocytes.
Chemokines and their receptors determine the distribution of leukocytes within tissues both in healthy and disease states. CXCR-4 and its ligand SDF-1 are found to be involved in the perivascular accumulation of T cells in rheumatoid arthritis. Mast cells are generally considered to be less mobile, residing within tissue sites. However, mast cells increase during inflammation, and are recognized to be important in regulating local neutrophil infiltration. Stimulation of human mast cells with SDF-1 induces a significant increase in intracellular calcium levels. In vitro, SDF-1 mediates dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayer.
SDF-1 is a chemoattractant for CD34(+) progenitor cells, both in vitro and in vivo, and SDF-1 and CXCR-4 are involved in homing of progenitor cells to bone marrow. Experiments indicated that SDF-1 is involved in hematopoiesis, and promotion of the proliferation of human CD34(+) cells purified from normal adult peripheral blood (PB). When CXCR-4 was expressed on PB CD34(+) cells, the amount of CXCR-4 on PB CD34(+) cells was found to be 10 times higher when CD34(+) cells were purified following overnight incubation. CXCR-4 overexpression is correlated with a primitive PB CD34(+) cell subset defined by a CD34(high) CD38(low) CD71(low)c-Kit(low)Thy-1(+) antigenic profile. The functional significance of CXCR-4 expression was ascertained by the promoting effect of SDF-la on cell cycle, proliferation, and colony formation. SDF-1 alone increases the percentage of CD34(+) cells in the S+G(2)/M phases and sustains their survival. In synergy with cytokines, SDF-1 increases PB CD34(+) and CD34(high)CD38(low) cell expansion and colony formation.
A variety of diseases require treatment with agents, which are preferentially cytotoxic to dividing cells. Cancer cells, for example, may be targeted with cytotoxic doses of radiation or chemotherapeutic agents. A significant side-effect of cancer therapy is the pathological impact of such treatments on rapidly dividing normal cells. These normal cells may for example include hair follicles, mucosal cells and the hematopoietic cells, such as primitive bone marrow progenitor cells and stem cells.
Hematopoietic cells that are uncommitted to a final differentiated cell type are defined herein as xe2x80x9cprogenitorxe2x80x9d cells. Hematopoietic progenitor cells possess the ability to differentiate into a variety of cell types directly or indirectly through a particular developmental lineage. Undifferentiated, pluripotent progenitor cells that are not committed to any lineage are referred to herein as xe2x80x9cstem cells.xe2x80x9d All hematopoietic cells can in theory be derived from a single stem cell, which is also able to perpetuate the stem cell lineage as daughter cells become differentiated.
Indiscriminating destruction of hematopoietic cells, such as stem, progenitor or precursor cells, can lead to a reduction in normal mature blood cell counts, such as leukocytes and red blood cells. A major impact on mature cell numbers may be seen particularly with neutrophils (neutropaenia) and platelets (thrombocytopenia), cells which naturally have relatively short half-lives. A decrease in leukocyte count, with concomitant loss of immune system function, may increase a patient""s risk of opportunistic infection. Neutropaenia resulting from chemotherapy may for example occur within two or three days of cytotoxic treatments, and may leave the patient vulnerable to infection for up to 2 weeks until the hematopoietic system has recovered sufficiently to regenerate neutrophil counts. A reduced leukocyte count (leukopenia) as a result of cancer therapy may become sufficiently serious that therapy must be interrupted to allow the white blood cell count to rebuild. Interruption of cancer therapy can in turn lead to survival of cancer cells, an increase in the incidence of drug resistance in cancer cells and ultimately in cancer relapse. There is accordingly a need for therapeutic agents and treatments, which facilitate the preservation or regeneration of hematopoietic cell populations in cases where the number of such cells has been reduced due to disease or to therapeutic treatments such as radiation and chemotherapy.
Bone marrow transplantation has been used in the treatment of a variety of hematological, autoimmune and malignant diseases. In conjunction with bone marrow transplantation, ex vivo hematopoietic (bone marrow) cell culture may be used to expand the population of hematopoietic cells, particularly progenitor or stem cells, prior to reintroduction of such cells into a patient. In ex vivo gene therapy, hematopoietic cells may be transformed in vitro prior to reintroduction of the transformed cells into the patient. In gene therapy, using conventional recombinant DNA techniques, a selected nucleic acid, such as a gene, may be isolated, placed into a vector, such as a viral vector, and the vector transfected into a hematopoietic cell, to transform the cell, and the cell may in turn express the product coded for by the gene.
The cell may then be introduced into a patient. Hematopoietic stem cells were initially identified as a prospective target for gene therapy. However, problems have been encountered in efficient hematopoietic stem cell transfection. There is accordingly a need for agents and methods, which may facilitate the proliferation of hematopoietic cells in ex vivo cell culture. There is also a need for agents that may be used to facilitate the establishment and proliferation of engrafted hematopoietic cells that have been transplanted into a patient.
A number of proteins have been identified as stimulators of hematopoietic cell proliferation (some of which are identified as hematopoietic growth factors). Cytokines involved in the induction of differentiation or proliferation of hematopoietic cells, particularly progenitor cells, include the following: G-CSF (granulocyte colony stimulating factor); LIF (leukemia inhibitory factor) and GM-CSF (granulocyte-macrophage colony stimulating factor).
The alpha Chemokine CXCR-4 and its ligand SDF-1 are postulated to be important in the development of the B-cell arm of the immune system. CXCR-4 is a critical coreceptor in support of viral entry by T-cell line tropic strains (X4) of the Human Immunodeficiency Virus Type 1 (HIV-1), viral variants, which predominate in some infected individuals in end stage disease. SDF-1 blocks X4-tropic HIV-1 infection of CD4+target cell in vitro, and allelic variants of the human gene encoding SDF-1 in vivo correlate with delayed disease progression. Thus, CXCR-4 may be an appropriate target for therapeutic intervention in acquired immunodeficiency syndrome (AIDS).
The localization of chronic lymphocytic leukemia (CLL) B-cells in bone marrow is not a adhesion phenomenon but a crucial step for their survival. SDF-1 produced by bone marrow stromal cells plays an important role in B-lymphocyte development and trafficking. It is found that Chemokine system SDF-1/CXCR-4 plays an important role in the accumulation of CLL B-cells.
Prostate neoplasm has a striking tendency to metastasize or xe2x80x9chomexe2x80x9d to bone. Metastases occur, when malignant cells escape from the primary tumor, penetrate and circulate through the bloodstream and subsequently arrest in the target tissues. It is found that metastatic prostate carcinoma utilizes the SDF-1/CXCR-4 pathway to localize to the bone marrow. Studies indicate that prostate cancers and perhaps other neoplasms (i.e. breast) may use the SDF-1/CXCR-4 pathway during their hematogenous spread to bone.
In various aspects, the invention provides methods for the use of chemokine-receptor-binding compounds (which may be chemokine receptor ligands such as chemokine receptor agonists or antagonist), and/or salts thereof, in treating chemokine mediated diseases or chemokine receptor mediated diseases, such as SDF-1 mediated diseases, or diseases mediated by chemokine receptors CXCR-4.
In some embodiments, the invention relates to methods of using a compound of formula (I), or a pharmaceutically acceptable salt thereof, to formulate a medicament for the treatment of a chemokine mediated disease state, or to treat such a disease: 
In some embodiments, ring A may be aromatic and may be heterocyclic with one or more heteroatoms selected from the group consisting of oxygen and nitrogen. In Formula I, Ring B may be aromatic or non-aromatic, may be heterocyclic with one or more heteroatoms selected from the group consisting of oxygen and nitrogen , xe2x80x9cxxe2x80x9d may for example represent a substitution at any position in ring B (in accordance with the presence or absence of hetroatom) where the substituents may be from the group consisting of hydrogen, hydroxyl, methoxy, carboxyl, esters (alkyl, phenyl or benzyl) alkyl, alkenyls, alkynyls, amino, amido, thio, thiazolo, imidazolo or may be fatty acids.
In alternative embodiments, R1 and R2 at each occurance may independently be selected from substituents having 50 or fewer atoms, wherein the substituent may be selected from the group consisting of: hydrogen, cyano, nitro, amino, sulfonyls, methoxy and fluoro; and combinations thereof.
In alternative embodiments, R3, R4, R5, R6, R7 and R8 at each occurance may independently be selected from substituents having 30 or fewer atoms, wherein the substituent may be selected from the group consisting of: H; substituted or unsubstitued alkyls, such as C1-5 alkyls; substituted or unsubstitued cycloalkyls, such as C3-5 cycloalkyls; substituted or unsubstitued alkenyls, such as C2-5 alkenyls; substituted or unsubstitued alkynyls, such as C2-6 alkynyls; substituted or unsubstitued aryls; such as benzyl and benzyl esters; substituted or unsubstitued heterocycles; hydroxyls; aminos; nitros; thiols; primary, secondary or tertiary amines; imines; amides; imidos, imidazoles; thiazoles; phosphonates; phosphines; carbonyls; carboxyls; silyls; ethers; thioethers; sulfonyls; sulfonates; selenoethers; ketones; aldehydes; esters; xe2x80x94CF3; xe2x80x94CN; amino acids, long chain amino acids, fatty acids and combinations thereof.
In formula I, xe2x80x9cXxe2x80x9d represents substitution in ring xe2x80x98Bxe2x80x99 at any position, where xe2x80x98nxe2x80x99 may be 0 or an integer from 1 to 4. xe2x80x9cYxe2x80x9d may be a variable group, representing xe2x80x98Oxe2x80x99 (oxygen) or xe2x80x98Nxe2x80x99 (nitrogen). xe2x80x9cZxe2x80x9d may be an sp2 carbon that may be part of a functional group as follows: 
For compounds of formula I, VI and VII, xe2x80x9cnxe2x80x9d may be 0 or an integer from 1 to 4 and xe2x80x9cnxe2x80x2xe2x80x9d may be 0 or an integer from 1 to 4.
In some embodiments, the chemokine may be selected from the group consisting of: SDF-1, and chemokines that bind to CXCR-4.
In various embodiments, the invention provides for the use of compounds of the invention in the treatment of diseases selected from the group consisting of inflammation, chronic and acute inflammation, arthritis, rheumatoid arthritis, osteoarthritis, ARDS, psoriasis, allograft rejection, chronic transplant rejection, asthma, atherosclerosis, mononuclear-phagocyte dependent lung injury, idiopathic pulmonary fibrosis, atopic dermatitis, chronic obstructive pulmonary disease, sickle cell disease, ulcerative colitis, septic shock, endotoxic shock, urosepsis, glomerulonephritis, lupus nephritis, thrombosis, graft vs. host disease, angiogenesis, NSCLC, human ovarian cancer, human pancreatic cancer, breast carcinoma, colon carcinoma, rectum carcinoma, lung carcinoma, oropharynx carcinoma, hypopharynx carcinoma, esophagus carcinoma, stomach carcinoma, pancreas carcinoma, liver carcinoma, gallbladder carcinoma, bile duct carcinoma, small intestine carcinoma, uterine carcinoma, kidney carcinoma, bladder carcinoma, urothelium carcinoma, female genital tract carcinoma, cervix carcinoma, uterus carcinoma, ovarian carcinoma, choriocarcinoma, gestational trophoblastic disease, male genital tract carcinoma, prostate carcinoma, seminal vesicles carcinoma, testes carcinoma, germ cell tumors, endocrine gland carcinoma, thyroid carcinoma, adrenal carcinoma, pituitary gland carcinoma, skin carcinoma, hemangiomas, melanomas, sarcomas, bone and soft tissue sarcoma, Kaposi""s sarcoma, tumors of the brain, tumors of the nerves, tumors of the eyes, tumors of the meninges, astrocytomas, gliomas, malignant gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, meningiomas, solid tumors arising from hematopoietic malignancies (such as leukomias, chloromas, plasmacytomas, and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia), solid tumors arising from lymphomas, non Hodgkin""s lymphoma (NHL), diseases relating to abnormal proliferation of hematopoietic cells, hematopoietic stemcytopenia after bone marrow transplantation, leukocytopenia, neutropenia, thromocytopenia, leukopenia, lymphopenia after chemotherapy, ex vivo gene therapy in bone marrow transplant and/or blood transufion, in the treatment of hematopoietic progenitor and stem cell proliferation and migration disorders, antiviral infections, HIV, AIDS, and neurodegenerative diseases such as Alzheimer, Parkinson""s, multiple sclerosis, disorder of bone metabolism such as osteoporesis.
In accordance with various aspects of the invention, CXCR4 antagonists may be used to treat hematopoietic cells, for example to increase the rate of hematopoietic stem or progenitor cellular multiplication, self-renewal, expansion, proliferation, or peripheralization. In various aspects, the invention relates to methods of promoting the rate of hematopoietic cell multiplication, which encompases processes that increase and/or maintain cellular multiplication, self-renewal, expansion, proliferation or peripheralization. This may for example be useful in some embodiments for in vitro hematopoietic cell cultures used in bone marrow transplantation, peripheral blood mobilization, or ex vivo expansion. CXCR4 antagonists may also be used therapeutically to stimulate hematopoietic cell multiplication, self-renewal, expansion, proliferation or peripheralization in vivo, for example in some embodiments involving human diseases such as a cancer or an autoimmune disease. The hematopoietic cells targeted by the methods of the invention may include hematopoietic progenitor or stem cells.
In alternative embodiments, CXCR4 antagonists may be used to treat a variety of hematopoietic cells, and such cells may be isolated or may form only part of a treated cell population in vivo or in vitro. Cells amenable to treatment with CXCR4 antagonists may for example include cells in the hematopoietic lineage, beginning with pluripotent stem cells, such as bone marrow stem or progenitor cells, lymphoid stem or progenitor cells, myeloid stem cells, CFU-GEMM cells (colony-forming-unit granulocyte, erythroid, macrophage, megakaryocye), B stem cells, T stem cells, DC stem cells, pre-B cells, prothymocytes, BFU-E cells (burst-forming unitxe2x80x94erythroid), BFU-MK cells (burst-forming unitxe2x80x94megakaryocytes), CFU-GM cells (colony-formng unitxe2x80x94granulocyte-macrophage), CFU-bas cells (colony-forming unitxe2x80x94basophil), CFU-Mast cells (colony forming unitxe2x80x94mast cell), CFU-G cells (colony forming unit granulocyte), CFU-M/DC cells (colony forming unit monocyte/dendritic cell), CFU-Eo cells (colony forming unit eosinophil), CFU-E cells (colony forming unit erythroid), CFU-MK cells (colony forming unit megakaryocyte), myeloblasts, monoblasts, B-lymphoblasts, T-lymphoblasts, proerythroblasts, neutrophillic myelocytes, promonocytes, or other hematopoietic cells that differentiate to give rise to mature cells such as macrophages, myeloid related dendritic cells, mast cells, plasma cells, erythrocytes, platelets, neutrophils, monocytes, eosinophils, basophils, B-cells, T-cells or lymphoid related dendritic cells.
In some embodiments, the invention provides methods of increasing the circulation of hematopoietic cells by mobilizing them from the marrow to the peripheral blood comprising administering an effective amount of a CXCR4 antagonist to hematopoietic cells of a patient undergoing autologous mobilization where hematopoietic stem/progenitor cells may be mobilized into the peripheral blood (1) during the rebound phase of the leukocytes and/or platelets after transient granulocytopenia and thrombocytopenia induced by myelosuppressive chemotherapy, (2) by hematopoietic growth factors, or (3) by a combination of both. Such treatment may for example be carried out so as to be effective to mobilize the hematopoietic cells from a marrow locus (i.e. a location in the bone marrow) to a peripheral blood locus (i.e. a location in the peripheral blood). Such treatments may for example be undertaken in the context of or for the clinical procedure of leukapheresis or apheresis. In alternative embodiments, CXCR4 antagonists may be used in ex vivo stem cell expansion to supplement stem cell grafts with more mature precursors to shorten or potentially prevent hematopoietic cell depletion, including conditions such as pancytopenia, granulocytopenia, thrombocytopenia, anemia or a combination thereof; to increase the number of primitive progenitors to help ensure hematopoietic support for multiple cycles of high-dose therapy; to obtain sufficient number of stem cells from a single marrow aspirate or apheresis procedure, thus reducing the need for large-scale harvesting of marrow of multiple leukopheresis; to generate sufficient cells from a single cord-blood unit to allow reconstitution in an adult after high-dose chemotherapy; to purge stem cell products of contaminating tumour cells; to generate large volumes of immunologically active cells with antitumour activity to be used in immunotherapeutic regimens or to increase the pool of stem cells that could be targets for the delivery of gene therapy.
In alternative embodiments, the invention provides methods to enrich CD34+progenitor cells which are utilized in bone marrow (BM) and peripheral blood (PB) stem cell transplantation, wherein the hematopoietic stem cell transplantation (HSCT) protocols may for example be utilized for the purpose of treating the following diseases (from Ball, E. D., Lister, J., and Law, P. Hematopoietic Stem Cell Therapy, Chruchill Livingston (of Harcourt Inc.), New York (2000)): Aplastic Anemia; Acute Lymphoblastic Anemia.; Acute Myelogenous Leukemia; Myelodysplasia; Multiple Myeloma; Chronic Lymphocytic Leukemia; Congenital Immunodeficiencies (such as Autoimmune Lymphoproliferative disease, Wiscott-Aldrich Syndrome, X-linked Lymphoproliferative disease, Chronic Granulamatous disease, Kostmann Neutropenia, Leukocyte Adhesion Deficiency); Metabolic Diseases (for instance those which have been HSCT indicated such as Hurler Syndrome (MPS I/II), Sly Syndrome (MPS VII), Chilhood onset cerebral X-adrenoleukodystrophy, Globard_cell Leukodystrophy).
In alternative embodiments, the invention relates to the use as CXCR4 antagonists of compounds of Formula I, with the exception of one or more of the compounds of formulae II or III or IV or V or VI or VII.